Some chemical substances, such as enzymes, provide catalysis of chemical action without undergoing permanent chemical change. These substances, known as catalysts, remained active only so long as their environment does not cause alteration to their molecular structure. Environmental changes such as pH, increase in temperature, metal ions attaching, and chemical inhibitors reduce catalytic activity of many of these substances. Often the presence of such factors are unpredictable.
For instance, by examining an appropriate enzyme, enzyme activity may be used to estimate a degree of cellular damage to the human body and to suggest a location where such damage may have occurred. Investigations of this type are particularly responsive to determination of cardiac, pancreatic, hepatic, and muscular and bone disorders.
In most kinetic reactions, the rate of conversion of a substrate to a product is a function of the level of the activity of the catalytic substance (enzyme) involved. Activity of this type is generally measured by measuring the absorption or transmittance of light through a solution, which specifically indicates the reduction in concentration of a substrate or the increase in concentration of a product. Transmittance of light can be converted by a logarithmic function to indicate light absorbance. Calculating the logarithm of measured light transmittance provides a linear function indicating absorbance. Absorbance indicates the rate of change in concentration of the substrate or product, which generally is a linear function with time for enzymatic reactions, and thus activity of a catalytic substance (enzyme).
Photometric measurement techniques are important since a common enzyme assay does not correctly measure the concentration of an enzyme in the sample. Rather, the assay monitors the amount of catalytic work or activity performed by the enzyme. To equate this activity measurement to enzyme quantity or concentration, the reaction conditions must be held constant and carefully controlled. Furthermore, these reaction conditions must be established and validated each time the assay is performed.
The study materials of a reaction are referred to as substrates which undergo a chemical change to form products to the reaction. In the presence of catalyzing enzyme, a reaction will proceed to form a product at a rate which depends on the concentration of the enzyme and other factors such as substrate concentration, temperature, and pH. It is important to initially determine that a proper solution mixture has been obtained to allow a reaction to proceed in the proper direction.
The activity of a specific enzyme can be quantitated even in complex mixtures containing other enzymes by measuring what the enzyme can do rather than by measuring the enzyme in terms of its mass or quantity. If substrate or product changes are measured by sensitive procedures, it is possible to relate these changes quantitatively to the activity of minute amounts of the associated enzyme. Thus the degree of catalytic activity can be utilized as a precise measurement of enzyme concentration.
Most commonly used is the rate of reaction assay. This method measures the rate at which a substrate is consumed or a product formed with respect to time, rather than the amount of a product or substrate in solution. The maximum rate of change of the substrate or product is directly related to enzyme activity. The maximum rate of conversion of substrate to product, or normally termed the rate of reaction, is easily determined through mathematical means.
The initial rate of an enzymatic reaction is directly proportional to the quantity of active enzyme present when substrate concentrations are at saturated levels and other environmentl variables are optimized and maintained at a constant value.
In some chemical reactions, several possible reaction pathways may exist. Catalysts such as an enzyme may favor one path over another resulting in a different yield of various reaction products, as compared with the uncatalyzed process. This is generally due to reduced energy levels required by a catalyzed process or pathway as compared to the uncatalyzed one. However, such processes often require a base energy level for fulfillment. Thus it may be difficult to ascert along which path a chemical reaction progressed until measurements of selected parameters are performed.
Catalyst (enzyme) activity measurements are accurate only if activity is measured under well defined experimental conditions. Thus the main need for working with enzymes is to define the conditions applicable to each measurement of a specific enzyme or reaction. And once the conditions are defined, a method of validating or assuring these conditions have been maintained is absolutely required.
For instance, a reaction may not proceed as expected due to a number of factors:
(1) concentrations of the reactants may not be as expected; PA1 (2) the age of the solution may be different from expected; PA1 (3) extreme values of pH may have irreversibly denatured the enzyme and thus reducing its ability to catalyze the reaction; PA1 (4) extreme values of temperature may have affected the reaction; PA1 (5) acids or bases may be formed as products from the reaction which may affect the continued process of the reaction.
These numerous factors show a great need for a method to validate the results obtained in measuring a catalyzed (enzymatic) reaction, in view of the number of problems which may cause inaccurate results.